Irradiation device and method for fluids especially for body fluids

ABSTRACT

A device suitable for use in the sterilization of a fluid such as a biological fluid or a fraction thereof, containing lymphocytes and/or micro-organisms, comprising a vessel having an inlet and an outlet and a passage which extends non-tortuously therebetween. A heat exchange device with a heat exchange surface is in substantially direct thermal contact with the interior of the passage. A temperature controller maintains the temperature of fluid in the passage below a temperature at which fluid components may form insoluble particles during irradiation. The passage has a wall which is substantially transparent to a lymphocyte and/or micro-organism inactivating radiation. The passage contains a static mixer device for thoroughly mixing the fluid so as to bring the whole of the fluid into an irradiation zone extending along and adjacent the passage walls and into contact with the heat exchange surface.

The present invention relates to the treatment of biological fluids,especially body fluids, and fractions thereof to inactivate selectedcomponents, e.g. lymphocytes, and microorganisms, including viruses andthe like, in human blood and in particular to a device suitable for usein such a procedure.

Large amounts of body fluids such as blood and plasma and variousfractions thereof are used in the treatment of patients suffering from avariety of conditions. Contamination of such fluids with various virusesand other microorganisms; however, can give rise to serious newconditions in the patients receiving these fluids and may even result intheir death.

Although it has been known for some time that ultra-violet (UV)irradiation can inactivate lymphocytes and viruses, this was not apractical procedure because of the very low UV transmissibility of bloodand hence the difficulty of ensuring a complete irradiation andinactivation. More recently we have considerably reduced this problem inour Patent No. GB 2200020 with the use of static mixers which provided avery thorough mixing of the fluid during irradiation thereby permittinga substantially even irradiation of the whole of the fluid.

We have now found, that fluids containing fibrinogen are susceptible toactivation and formation of more or less large particles of polymericfibrin. Such particles can moreover form around viruses and othermicroorganisms and thus screen them from the UV radiation therebypreventing inactivation thereof, and thus seriously risking the healthof the recipient of the treated fluid. This fibrinogen activation can bereadily triggered by mechanical stress e.g. shear forces present inmixing and by heat which can readily occur locally during irradiation.Insoluble particles of material can also be formed by thermal and/ormechanical denaturation of other proteinaceous components.

Such problems also arise with conventional sterilization of human bloodproducts which generally involves incubation thereof at a temperature ofthe order of 78° C. for an extended period of time of perhaps 48 to 72hours. This procedure further has the disadvantages of being relativetime consuming and occupying substantial amounts of relatively largescale apparatus and may result in substantial loss of potency.

It is an object of the present invention to avoid or minimize one ormore of the above disadvantages.

We have now found that by carefully controlling the temperature of thefluid and preventing any localized heating thereof, formation ofparticles which can screen viruses and other microorganisms frominactivating radiation, can be substantially prevented.

In one aspect the present invention provides a device suitable for usein use in the sterilization of a fluid, which is a biological fluid or afraction thereof, containing lymphocytes and/or micro-organisms, whichdevice comprises a vessel having an inlet and an outlet and a passagemeans extending substantially directly and non-tortuously therebetween,said passage means having a heat exchange device with a heat exchangesurface in substantially direct thermal contact with the interior of thepassage means, and a temperature control means formed and arranged formaintaining the temperature of fluid in the passage below a temperatureat which fluid components may form insoluble particles duringirradiation, and said passage means having wall means substantiallytransparent to a lymphocyte and/or microorganism inactivating radiation,said passage means containing a static mixer device formed and arrangedfor thoroughly mixing the fluid in use of the device, so as to bringsubstantially the whole of the fluid into an irradiation zone extendingalong and in substantially direct proximity to said wall means duringpassage between said inlet and said outlet and into contact with saidheat exchange surface, whereby in use of the device substantially thewhole of a body of said fluid passed through said vessel may be exposedto a similar substantial level of irradiation whilst maintaining it at asafe temperature.

Thus with a device of the present invention a particularly uniformtreatment of the fluid with respect to both irradiation and temperaturethereof may be achieved thereby avoiding on the one hand under-exposureto inactivating radiation whether as a result of screening by anexcessive depth of soluble fluid components or by enveloping insolublematerial formed by more or less direct thermal denaturation of fluidcomponents or as a result of thermally and/or mechanically triggeredreactions (such as fibrinogen activation), as well as avoiding localizedoverheating induced by the irradiation which can result in reducedinactivation and/or increased degradation, thereby on the one handmaximizing inactivation of lymphocytes (where required) and/orundesirable microorganisms and on the other hand minimizing denaturationand degradation of useful fluid components.

It will be appreciated that various forms of heat exchange device may beused including solid state devices such as Peltier effect devices.Conveniently though there is used a heat exchange device wherein iscirculated a heat exchange fluid (e.g. gas, liquid, or liquid mixed withgas and/or frozen liquid) as this generally facilitates more precisecontrol of the biological fluid temperature.

Conveniently there is used an annular form of vessel with an outer wallsubstantially transparent to lymphocyte and/or microorganisminactivating radiation and an inner wall constituting said heat exchangesurface, the latter preferably being of a generally inertphysiologically compatible material with high thermal conductivity e.g.stainless steel. Any suitable heat exchange fluid may be used e.g.water. Where a heat exchange fluid is used then the temperature of thismay be controlled in various ways remotely from said heat exchangesurface in the vessel e.g. using a solid state heat exchanger such as aPeltier-effect heat pump or a refrigeration coil etc.

Various forms of temperature control means may be used. In general thereis used a variable rate cooling device provided with a controller forvarying the cooling rate according to an input from a temperature sensormeans disposed in thermal connection with at least one of the fluidpassage means, the heat exchange surface, and a heat exchange fluidpassage in said heat exchange device.

It will be appreciated that the “safe” temperature limits for avoidingdenaturation and/or other thermally triggered reaction may vary from onebiological fluid or fraction thereof to another, and also depending onthe application of the fluid, and conveniently there is used atemperature control means which allows for the fluid temperature to bemaintained at a plurality of different values as required. In generalfor any body fluids containing fibrinogen the temperature is desirablymaintained at not more than 37° C., preferably from −5 to 37° C.,advantageously from −5 to 19° C.

In a preferred aspect of the invention the device includes at least onemicroorganism inactivating radiation source mounted in more or lessclosely spaced proximity to said transparent wall means (for theavoidance of doubt it should be noted that references to the transparentwall means merely indicates substantial transmission of the inactivatingradiation which may or may not be accompanied by significanttransparency at other wavelengths e.g. visible light). The mounting ofthe radiation source is generally arranged to minimize undesired heatingof the transparent wall means and biological fluid in contact therewithwhilst maximizing the radiation intensity in the irradiation zone.Depending on the source used this generally positioned at from 1.5 to 5mm from the transparent wall.

Various lymphocyte and/or microorganism inactivating radiations may beused, though UV is generally preferred, especially UV radiation having awavelength range from 100 to 400 nm preferably from 200 to 350 nm, forexample UVA at approximately 320 to 500 nm. UVB at approximately 310 nmand UVC at approximately 254 nm.

Suitable UV lamp sources are readily available commercially. Particularlamp sources which may be mentioned include those available from GTESylvania Ltd. of Charlestown, Shipley, West Yorkshire. Thorn EMI ofEnfield, Middlesex and Philips Lighting of Croydon, Surrey, all inUnited Kingdom.

It should also be noted that the present invention also includes withinits scope indirect inactivation of microorganism whereby aphotoactivatable drug is incorporated in the fluid, said drug beingconverted from a non-activating form into a microorganism inactivatingform by U.V. irradiation. One example of a photoactivatable drug of thistype that may be mentioned is a psoralen e.g. 8—methoxy psoralen whichupon exposure to U.V.—A radiation of 320 to 400 nm wavelength becomescapable of forming photoadducts with DNA in lymphocytes therebyinactivating these.

Where UV radiation is used to effect inactivation then the vessel sidewall means may be made of various UV—transparent materials including forexample silica and other UV—transparent glasses such as those availableunder the Trade Names Spectrosil and Vitreosil; silicones; celluloseproducts such as Cellophane (Trade Name); and plastics materials such aspolytetrafluoroethylene (PTFE), fluoroinatedethylenepropylene (FEP), andpreferably low density polyethylene (LDPE) or polyvinyl chloride (PVC).Other activating radiations that may be used include microwave radiationused in conjunction with e.g. a glass or ceramic vessel wall; infra-redradiation used in conjunction with e.g. a quartz vessel wall; ultrasoundradiation used in conjunction with e.g. a stainless steel vessel wall.

The duration of irradiation required will depend on various factors suchas the intensity, disposition, and number of sources used, thetransmission characteristics of the vessel side wall material, thevessel configuration and hence the mixing efficiency therein and thesurface area of the thin layer of fluid adjacent the vessel side wall,the length of the passage means in the vessel and the flow-rate of thefluid being treated, and hence the residence time of the fluid in theirradiation zone, as well as the nature of the fluid itself. Therequired duration may however be readily determined by simple trial anderror using suitable techniques known in the art for assessinginactivation of the relevant microorganisms and further details areprovided hereinbelow. In general the residence time in the vessel willconveniently be in the range from 5 seconds to 30 minutes, preferablyfrom 30 seconds to 10 minutes, e.g. 2 minutes, and the vessel side wallmaterial and thickness and the radiation sources are chosen andarranged, to provide an effective inactivating dosage of U.V. radiationwithin such a period.

It will moreover be appreciated that the required irradiation time canbe achieved in a number of different ways including one or more of thefollowing: use of vessels with irradiation zones of different length,varying the flow rate of the fluid, using a plurality of devices inseries, and recycling the fluid through the device(s) a number of times,though generally it is highly desirable that the inactivation treatmentsystem is designed so that the required level or irradiation is achievedin a single pass, especially where the inactivation treatment system isincorporated in a production line for the manufacture of variousproducts e.g. IgG, Factor VIII etc. etc.

Whilst it is a particular advantage of the present invention thatdenaturation of useful body fluid components is minimized, there canadvantageously be included in the body fluid one or more protectantssuch as rutin, Ascorbic acid (ca 1 mM), or Quercetin (ca 0.2 mM), whichreduce still further any possible denaturation or degradation of usefulcomponents.

It will also be understood that the degree of mixing required to achievecomplete irradiation will depend on various factors such as thetransmissibility of the fluid to the inactivating radiation and thetotal depth of fluid in the vessel from the wall through which radiationis received. In general the lower the transmissibility and the greaterthe fluid depth, the greater will be the number of mixer elements andmixing stages required.

Further preferred features and advantages of the invention will appearfrom the following detailed description given by way of examples andillustrated with reference to the accompanying drawings in which:

FIG. 1 is a partly schematic partly sectioned view of an irradiationapparatus of the present invention.

FIG. 1 shows an apparatus 1 comprising a vessel 2 in the form of acylindrical tube 3 of quartz or other UV—transmissible material with aninlet 4 and an outlet 5, with an axially extending static mixer device 6provided with temperature control means 7. In more detail the staticmixer device 6 comprises an axially extending series of angularly offsethelical “screw” elements 8 defining pairs of flow paths which aredivided equally and mixed at the junctions 9 between successive elements8 thereby providing a degree of mixing which increases exponentiallywith the number of elements used.

The “screw” elements 8 are mounted on a hollow core 10 which defines aheat exchange fluid passage 11 forming part of the temperature controlmeans 7. In more detail the temperature control means 7 comprises a heatexchange fluid circuit 12 provided with pump means 13 for circulatingthe heat exchange fluid 12 a therethrough and a Peltier-effect heatexchange device 14 provided with a control means 15 which has atemperature sensor 16 mounted inside the vessel 3 for monitoring thetemperature of the fluid undergoing irradiation. The control means 15 isformed and arranged for controlling the rate of cooling supplied so asto maintain a desired fluid temperature. This may be a fixed value, ormore conveniently the control means 15 may be provided with useroperable input means for varying the desired temperature setting.

The core 10 (and desirably also the screw elements 8) are of an inertphysiologically acceptable thermally conductive material such asstainless steel in order to facilitate efficient thermal transferbetween the fluid being treated 17 and the heat exchange circuit 7thereby to control the fluid temperature closely within relativelynarrow limits so as to on the one hand maximize the efficiency of thesterilization/inactivation treatment and on the other hand to minimizeany undesired denaturation or degradation of useful fluid components.

Irradiation 20 is effected by means of a plurality of UVC-emittingfluorescent tubes 21 extending parallel to and closely spaced from thevessel 3 and angularly distributed therearound. Advantageouslyreflectors 22 are provided to help concentrate the radiation 20 onto thevessel. The irradiation chamber may also be cooled by a fan 23. Thevessel 3 is made of quartz in order to maximize transmission of theradiation 20 into the fluid 17 being treated and has diameter ofapproximately 20 mm, and a mixer 6 with a length of 300 mm and 10elements.

The pump means 13 advantageously is provided with a flow rate controllerin order to vary the flow rate of the fluid 12 a to adjust the residencetime of the fluid in the vessel 3 in the irradiation zone 19 and also tominimize denaturation or degradation of useful fluid components arisingfrom mechanical stress in and around the static mixer 6. In generalthere may be used a flow rate of the order of 1 cm/sec to 100 cm/secpreferably 2 cm/sec to 50 cm/sec, desirably from 5 to 20 cm/sec.

It will be appreciated that the vessel 3 and mixer 6 may be found andarranged so that complete irradiation may be achieved with a single passof the fluid through the vessel. Alternatively though a plurality ofpasses may be used to achieve full irradiation.

Use of the apparatus will be further explained in the followingillustrative example.

EXAMPLE 1 Treatment of Human Plasma

Irradiation was carried out using an irradiation device having four 500mm long UVC light sources distributed around 6 mm internal diameter PTFEtube containing a 34 cm long static mixer of the type shown in FIG. 1with 48 screw elements. The fluid was circulated through the quartztube, and a cooling device mounted in series therewith, at a flow rateof 100 ml/min which corresponded to an irradiation time of approximately6.2 seconds for each passage. The fluid was circulated until a totaleffective irradiation time of approximately 100 seconds was achieved andthe temperature thereof maintained at around 6.5° C.

Using plasma samples (200 ml) into which has been introduced abacteriophage virus (2 ml) selected from: X174 and MS-2 (single-strandDNA and RNA respectively); T4 (double-strand DNA) and PR7772 (doublestrand DNA, enveloped), a virus kill in the region of 5-6 logs (i.e.over 99.999%) was achieved. At the same time the coagulation factoractivity of key components of the plasma was substantially maintained asfollows:

Factor CIII:C 57.3 ± 4.2% Factor V 38.8 ± 10.4% Fibrinogen 63.5 ± 4.2%APTT 18.5 ± 3.4%

In a further experiment rutin (1.6 mM) was introduced into the plasma asa protectant and the retained coagulation factor activity was increasedto over 85% whilst maintaining the virus inactivation level.

EXAMPLE 2

Using substantially similar procedures a human immunoglobulinpreparation (150 g of IgG per litre) containing MS-2 (1.5 g) wassubjected to an effective irradiation time of 300 seconds.

A virus inactivation level of 4.8 logs was achieved whilst aggregateformation increased from an initial level of 7.0% to only 7.6%.

Sterilization of the blood is monitored by one or more of the followingprocedures:

(a) Separation of lymphocytes, culture and subsequent dosage withtritiated thymidine and subsequent liquid scintillation counting.

(b) Separation of lymphocytes, culture and examination by electronmicroscope.

(c) Separation of lymphocytes and observation of response to tissuestains.

(d) Culture of bacteria by standard laboratory methods.

(e) Growth of viruses by standard laboratory methods.

(f) Study of Protozoans by light and electron microscopy and by in vivopassage in an animal species.

(g) Study of biological behaviors of Blood Platelets by standard invitro hematological techniques e.g. behavior in an agregometer and afterexposure to collagen, ATP etc.

What is claimed is:
 1. A device suitable for use in the sterilization ofa fluid, which is a biological fluid or a fraction thereof, containinglymphocytes and/or micro-organisms, which device comprises a radiationsource operable to produce a lymphocyte and/or micro-organisminactivating radiation, a vessel having an inlet and an outlet and apassage means extending substantially directly and non-tortuouslytherebetween, said passage means having a heat exchange device with aheat exchange surface extending along and in substantially directthermal contact with the interior of the passage means, and atemperature control means formed and arranged for maintaining thetemperature of fluid in the passage below a temperature at which fluidcomponents may form insoluble particles during irradiation, and saidpassage means having wall means substantially transparent to saidlymphocyte and/or micro-organism inactivating radiation, said passagemeans containing a static mixer device extending therealong and formedand arranged for thoroughly mixing the fluid in use of the device, so asto bring substantially the whole of the fluid into an irradiation zoneextending along and in substantially direct proximity to said wall meansduring passage between said inlet and said outlet and into contact withsaid heat exchange surface, whereby in use of the device substantiallythe whole of a body of said fluid passed through said vessel may beexposed to a similar substantial level of irradiation whilst maintainingit at a safe temperature.
 2. A device (1) as claimed in claim 1 whereinsaid heat exchange device (14) is a solid state Peltier-effect device.3. A device (1) as claimed in claim 1 wherein said heat exchange device(14) comprises a conduit means (10) for the passage of a heat exchangefluid (12 a) through the interior of said static mixer device (6) and insubstantially direct thermal contact with an external surface of saidstatic mixer device which constitutes said heat exchange surface of saidheat exchange device.
 4. A device (1) as claimed in claim 3 wherein saidvessel (2) has an annular form with an outer wall substantiallytransparent to a lymphocyte and/or microorganism inactivating radiation(20) and an inner wall constituting said heat exchange surface (10). 5.A device (1) as claimed in claim 4 wherein the temperature of said heatexchange fluid (12 a) is controlled (15) remotely from said heatexchange surface (10) in the vessel (2) by means of a solid state heatexchanger (14).
 6. A device (1) as claimed in claim 4 wherein saidtemperature control means (7) is in the form of a variable rate coolingdevice provided with a controller (15) for varying the cooling rateaccording to an input from a temperature sensor means (16) disposed inthermal connection with at least one of said fluid passage means (3),said heat exchange surface (10), and the heat exchange fluid passage(11) in said heat exchange device (14).
 7. A device (1) as claimed inclaim 1 for use in the sterilization of a body fluid containingfibrinogen wherein said temperature control means is formed and arrangedto maintain the temperature of said body fluid (17) at a temperature inthe range of from −5° C. to +37° C.
 8. A device (1) as claimed claim 1which includes at least one lymphocyte and/or microorganism inactivatingradiation source (21) mounted in more or less closely spaced proximityto said transparent wall means (3).
 9. A device as claimed in claim 8wherein said lymphocyte and/or microorganism inactivating radiationsource is an ultra violet radiation source having an ultra violetradiation wavelength in the range of from 200 to 350 nm.
 10. A device asclaimed in claim 9 wherein said device is formed and arranged so as tobe substantially free of any obstruction between said transparent wallmeans and said ultra violet radiation source.
 11. A device (1) asclaimed claim 1 wherein said side wall means (3) of said vessel (2) ismade of substantially ultraviolet-transparent materials selected fromthe group including UV-transparent glasses, silicone, celluloseproducts, and plastics materials.
 12. A device (1) as claimed in claim 1wherein there may be used inactivating radiation and vessel wallmaterial combinations selected from the group including microwaveradiation used in conjunction with glass; infra-red radiation used inconjunction with a quartz vessel wall; or ultra sound radiation used inconjunction with a metal vessel wall.
 13. A device (1) as claimed inclaim 1 wherein there are provided reflectors (22) spaced around thevessel (2) formed and arranged to concentrate the radiation (20) ontosaid vessel.
 14. A method of sterilizing a biological fluid (17) orfraction thereof, containing lymphocytes and/or microorganismscomprising the steps of: providing a device (1) according to claim 1;providing a lymphocyte and/or microorganism inactivating radiationsource (21) in more or less closely spaced proximity to said transparentwall means of said device (1); passing said fluid (17) through saidpassage means (3) of said device (1) so that the whole of a body of saidfluid (17) is exposed to a similar substantial level of lymphocyteand/or microorganism inactivating irradiation; and operating saidtemperature control means (7) of said device (1) so as to maintain thetemperature of the fluid (17) in the passage means (3) below atemperature at which fluid (17) components may form insoluble particlesduring irradiation.
 15. A method as claimed in claim 14 which includesthe step, prior to passing said fluid (17) through said passage means(3), of incorporating into the fluid (17) to be sterilized aphotoactivatable drug, said drug being convertible from a non-activatedform into a lymphocyte and/or microorganism-inactivating form byradiation (20).
 16. A method as claimed in claim 14 which includes thestep, prior to passing said fluid (17) through said passage means, ofincorporating into the fluid (17) to be sterilized at least oneprotectant to reduce further any possible denaturation or degradation ofuseful fluid components.
 17. A method as claimed in claim 14 wherein theresidence time of fluid (17) in said vessel (2) of said device (1) is inthe range of from 30 seconds to 10 minutes.
 18. A device as claimed inclaim 1 wherein said static mixer device comprises an axially extendingseries of angularly offset helical screw elements defining pairs of flowpaths which are divided equally and mixed at junctions betweensuccessive elements.
 19. A device as claimed in claim 18 wherein saidscrew elements are mounted on a hollow core which defines a heatexchange fluid passage.
 20. A device as claimed in claim 19 wherein saidcore and screw elements are of an inert physiologically acceptablethermally conductive material.
 21. A device as claimed in claim 20wherein said thermally conductive material is stainless steel.